CN102680442B - A method for label-free fluorescent detection of trypsin - Google Patents

Abstract

A method for detecting trypsin using unmarked fluorescence belongs to the technical field of interdisciplinary subjects of material, biology, and analytical chemistry, and particularly relates to the method for detecting the trypsin based on disassembly of supramolecular assemblies under enzymatic hydrolysis. The method is characterized by including: firstly, determining critical micellar concentration of a surfactant; secondly, forming the supramolecular assemblies with the surfactant and polyelectrolyte with opposite charges through hydrophobic and electrostatic interaction, and embedding hydrophobic dyes serving as fluorescent probes into the inner cavities of the supramolecular assemblies; thirdly, adding enzyme which can lead to hydrolysis in the polyelectrolyte so as to lead the disassembly of the supramolecular assemblies and release the embedded hydrophobic dyes, and detecting enzyme activity by lowering fluorescence intensity. The method is simple to prepare, low in cost, real-time in detection, and wide in application prospect in fields such as diagnosis and detection of biomolecules, and biosensors.

Description

A method for label-free fluorescent detection of trypsin

technical field

The invention discloses a method for detecting trypsin with unlabeled fluorescence, which belongs to the technical field of interdisciplinary materials, biology, and analytical chemistry, and specifically relates to a method for realizing trypsin detection based on supramolecular assembly disassembly under enzyme-catalyzed hydrolysis The technical solution of the method.

Background technique

Trypsin is one of the most important digestive enzymes in protein decomposition. The secretion, activation, inhibition and circulation imbalance of trypsinogen can lead to acute or chronic pancreatic diseases, such as pancreatic cancer. Pancreatic cancer is the most malignant tumor with the worst prognosis. The 5-year survival rate of patients is generally less than 5%. About 35,000 cases of pancreatic cancer are caused by the imbalance of serine proteases, especially trypsin, every year. In addition, trypsin not only acts as a digestive enzyme, but also restricts the decomposition of precursors of other enzymes such as chymotrypsinogen, carboxypeptidase, and phospholipase, and activates them.

Currently developed methods for the determination of trypsin mainly include UV spectrophotometry, colorimetry, fluorescence, immunoassay, radioactive element labeling, mass spectrometry (MS), liquid chromatography (HPLC), electrophoresis, etc. Ultraviolet and colorimetric methods are simple to operate, but have low sensitivity and poor reproducibility. Immunization method has high sensitivity, as described in patent 201020245770.x, but the cost is high due to the use of monoclonal antibodies, and certain operating techniques are required. The radioactive element labeling method needs to label the substrate, which is complicated to prepare and has certain safety problems and so on. Mass spectrometry instruments are expensive and require high purity of samples, while chromatography and electrophoresis require relatively complicated operations and long detection times.

Compared with these methods, the fluorescence method is easy to operate, has high sensitivity, can realize real-time monitoring, and has the potential of clinical application. However, the general fluorescence method requires a fluorescently labeled substrate, which is complicated to prepare and increases the cost, which limits its application. In recent years, label-free methods have attracted attention. The currently reported methods for label-free fluorescence detection mainly use water-soluble fluorescent conjugated polymers and new aggregation-induced luminescent molecules. The synthesis and separation of these molecules are still relatively complicated. Therefore, it is of great significance to establish a reagent that is easy to obtain and to prepare a simple fluorescence detection method. Through searching, there is no method for label-free fluorescent detection of trypsin.

Contents of the invention

The purpose of the method for detecting trypsin with unlabeled fluorescence is to overcome the deficiencies of the prior art and provide a method for detecting trypsin with unlabeled fluorescence that is simple, fast, sensitive and does not require organic synthesis.

A method for detecting trypsin with unlabeled fluorescence of the present invention is characterized in that it is a method for realizing trypsin detection based on supramolecular assembly disassembly under enzyme-catalyzed hydrolysis, and its specific steps are:

Ⅰ Preparation of organic solution of fluorescent probe:

Use hydrophobic dyes as fluorescent probes, dissolve the hydrophobic dyes in organic solvents such as commercially available spectrum pure acetone, chloroform or methanol, and prepare an organic solution with a concentration of 10 ^-3 ~ 10 ^-6 mol L ^-1 fluorescent probes;

Ⅱ Determination of critical micelle concentration CMC of surfactant:

Dissolve the surfactant in a phosphate buffer solution with a concentration of 10 mM and pH 8.0 to prepare a gradient solution with a concentration ranging from 1 mol L ^-1 to 10 ^-7 mol L ^-1 , and use a micro-injector to pipette step Ⅰ to prepare The organic solution of the fluorescent probe was added to the gradient solution, so that the final concentration of the probe in the gradient solution was 10 ^-8 ~10 ^-6 mol L ^-1 , treated with 40 ~ 100kHz exposure ultrasonic wave for 10 ~ 60 minutes, and let it stand After 0.5 to 12 hours, perform fluorescence spectrum detection to measure the critical micelle concentration CMC of the surfactant;

Ⅲ Preparation of supramolecular assembly solution:

According to the critical micelle concentration CMC of the surfactant obtained in step Ⅱ, the concentration of the surfactant is determined to be 0.1~1.6 CMC, and the oppositely charged polyelectrolyte is dissolved in a phosphate buffer solution with a concentration of 10 mM and a pH of 8.0. Polyelectrolyte solution: According to the charge ratio of polyelectrolyte and surfactant being 1:1, add an equal volume of polyelectrolyte solution to the surfactant solution, and the surfactant and the oppositely charged polyelectrolyte form a superhydrophobic and electrostatic interaction. The molecular assembly is uniformly mixed, and left to stand for 10 to 15 minutes to obtain a supramolecular assembly solution, and the weight percent concentration of the supramolecular assembly solution is 0.01 to 0.2%;

Ⅳ Preparation of test solution:

Pipette the fluorescent probe organic solution prepared in step I again with a microsampler, and add them to the supramolecular assembly solution obtained in III, so that the final concentration of the probe in the supramolecular assembly solution is 10 ^-8 ~ 10 ^-6 mol L ^-1 , treated with 40-100kHz exposure ultrasonic treatment for 10-60 minutes, and obtained the test solution after standing for 0.5-12 hours;

Ⅴ Determination of catalytic hydrolysis time:

Incubate the test solution obtained in step IV for 10-15 minutes at 35-37°C, dissolve trypsin in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and add the same volume of trypsin buffer solution to the test solution , using only buffer solution as a blank control, the final concentration of trypsin ranges from 0.01 to 100 μg mL ^-1 , and the fluorescence spectrum tracking detection is carried out at 35 to 37°C, and the ratio of the decrease in fluorescence emission peak intensity is plotted against time, and The time range for the linear decrease of fluorescence emission peak intensity under different concentrations of trypsin-catalyzed hydrolysis conditions;

Ⅵ Determination of trypsin activity:

According to the time range obtained in step V, the maximum value of the common linear time range under each concentration condition was taken, and the ratio of the decrease in fluorescence emission peak intensity was plotted against the concentration of trypsin added to obtain a detection standard curve.

The above-mentioned method for label-free fluorescent detection of trypsin is characterized in that the hydrophobic dye is commercially available pyrene, Nile red, coumarin 481, Sudan red or fluoroboron dye BODIPY with a purity higher than 98%. BODIPY refers to BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPY TR or BODIPY 630/650.

The above-mentioned method for label-free fluorescent detection of trypsin is characterized in that the surfactant is a negatively charged alkyl sulfate or alkyl sulfonate, and the alkyl sulfate is commercially available analytically pure dodecyl Sodium sulfate, sodium tetradecyl sulfate, or sodium cetyl sulfate; alkylsulfonates are commercial analytical grade sodium dodecyl sulfate, sodium tetradecyl sulfate, or sodium cetyl sulfate .

The above method for label-free fluorescent detection of trypsin is characterized in that the polyelectrolyte is a polypeptide that can be catalyzed and hydrolyzed by trypsin, and the polypeptide is commercially available with a purity higher than 99% and a length of 10-16 positively charged Arginine sequence Arg ₁₀ ~ Arg ₁₆ .

Compared with the prior art, a method for label-free fluorescent detection of trypsin activity of the present invention has beneficial effects:

1) Detection is carried out through the disassembly of supramolecular assemblies, which does not require complex organic synthesis and immobilization processes, and is simple to prepare, low in cost, and pollution-free;

2) Fluorescent dyes are embedded in the supramolecular assembly, and detected by fluorescence method, which has high sensitivity, short detection time, and easy real-time detection.

Description of drawings

Fig. 1 is the critical micelle concentration determination curve of surfactant sodium dodecyl sulfate (SDS);

Figure 2 shows the differences in the assembly behaviors of surfactant sodium dodecyl sulfate (SDS) and polypeptides of different lengths (Arg ₁₀ and Arg ₆ ).

Detailed ways

Using a hydrophobic dye as a fluorescent probe, the critical micelle concentration of the surfactant was firstly determined, and then below the critical micelle concentration of the surfactant, the surfactant and the oppositely charged polyelectrolyte formed a supramolecular assembly through hydrophobic and electrostatic interactions The hydrophobic dye is embedded in the lumen of the assembly, and finally an enzyme that hydrolyzes the polyelectrolyte is added to catalyze the hydrolysis of the high-molecular-weight polyelectrolyte into a low-molecular-weight polyelectrolyte, which induces the disassembly of the assembly and makes the assembly lumen-embedded The fluorescent probe is released, and the detection of enzyme activity is realized through the reduction of fluorescence intensity. The following embodiments are further descriptions of the present invention, rather than limiting the scope of the present invention.

Implementation mode 1:

Ⅰ Preparation of organic solution of fluorescent probe:

Using pyrene as a fluorescent probe, dissolve pyrene in acetone, prepare an acetone solution with a concentration of 10 ^-2 mol L ^-1 pyrene in a 10 mL volumetric flask, and further dilute to 10 ^-5 mol L ^-1 with acetone;

Ⅱ Determination of critical micelle concentration CMC of surfactant:

Sodium dodecyl sulfate SDS was dissolved in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and a solution of 10 ^-2 mol L ^-1 was prepared in a 10 mL volumetric flask, and further diluted with buffer solution, Obtain a gradient solution with a concentration range of 1mol L ^-1 ~10 ^-7 mol L ^-1 ;

Use a microsampler to pipette the organic solution of the fluorescent probe prepared in step I, and add it to the prepared gradient solution, so that the final concentration of the fluorescent probe in the gradient solution is 5×10 ^-7 mol L ^-1 . Ultrasonic treatment was performed at 100 kHz for 10 minutes, and the fluorescence excitation spectrum was detected after standing for 0.5 hour.

The ratio of the fluorescence excitation spectrum I ₃₃₈ /I ₃₃₃ was plotted against the logarithmic value of the surfactant concentration, and an s-shaped curve was obtained by fitting. The concentration corresponding to the first abrupt point of the curve was the critical micelle concentration of the surfactant. The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Determine the surfactant concentration as 0.6 CMC, dissolve Arg ₁₀ in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and add an equal volume of Arg ₁₀ buffer solution according to the charge ratio of polyelectrolyte to surfactant at 1:1 , the surfactant and the oppositely charged polyelectrolyte construct a supramolecular assembly through hydrophobic and electrostatic interactions, mix evenly, and let stand for 10 minutes to obtain an assembly solution, the weight percent concentration of which is 0.075%;

Ⅳ Preparation of test solution:

Pipette the fluorescent probe organic solution prepared in step I again with a microsampler, and add them to the assembly solution obtained in III, so that the final concentration of the fluorescent probe in the assembly solution is 5×10 ^-7 mol L ^{- 1.} Ultrasonic treatment at 100kHz for 10 minutes, and after standing for 0.5 hours, the test solution was obtained;

Ⅴ Determination of catalytic hydrolysis time:

Incubate the test solution obtained in step IV at 35°C for 10 minutes, dissolve trypsin in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and add the same volume of trypsin with different concentrations to the test solution to add The buffer solution was used as a blank control, the concentration ranged from 0.01 to 100 μg mL ^-1 , and the fluorescence spectrum tracking detection was carried out at 35°C. The ratio of the decrease of the fluorescence emission peak intensity was plotted against the time, and the fluorescence emission under different concentrations of trypsin catalyzed hydrolysis conditions was obtained. The time range in which the peak intensity decreases linearly;

Ⅵ Determination of trypsin activity:

According to the time range obtained in step V, the maximum value of the common linear time range under each concentration condition was taken, and the ratio of the decrease in fluorescence emission peak intensity was plotted against the concentration of trypsin added to obtain a detection standard curve.

The ratio of fluorescence intensity decrease is (II ₀ )/I ₀ ×100 %, I ₀ represents the fluorescence intensity at the fluorescence emission peak of the blank control with only buffer solution added, and I represents the fluorescence intensity at the emission peak after adding different concentrations of trypsin.

The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment: under the same test conditions, the same concentrations of alkaline phosphatase, lysozyme, glucose oxidase, and trypsin were added to the assembly solution, and the fluorescence spectrum was detected after incubation at 37°C for half an hour.

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Embodiment 2

Ⅰ Preparation of organic solution of fluorescent probe:

Using Nile Red as a fluorescent probe, dissolve Nile Red in methanol, prepare a methanol solution with a concentration of 10 ^-1 mol L ^-1 Nile Red in a 10 mL volumetric flask, and further dilute to 10 ^-3 mol with methanol L ^-1 ;

Ⅱ Determination of critical micelle concentration CMC of surfactant:

Sodium tetradecyl sulfate was dissolved in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and a solution of 10 ^-2 mol L ^-1 was prepared in a 10 mL volumetric flask, and further diluted with buffer solution to obtain Gradient solution with a concentration range of 1mol L ^-1 ~10 ^-7 mol L ^-1 ;

Use a microsampler to pipette the organic solution of the fluorescent probe prepared in step I, and add it to the prepared gradient solution, so that the final concentration of the fluorescent probe in the gradient solution is 10 ^-6 mol L ^-1 . Ultrasonic treatment was performed at 70 kHz for 30 minutes, and the fluorescence excitation spectrum was detected after standing for 1 hour.

The emission peak intensity of the fluorescence emission spectrum is plotted against the logarithmic value of the surfactant concentration, and an s-shaped curve is obtained by fitting, and the concentration corresponding to the first mutation point of the curve is the critical micelle concentration of the surfactant. The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Fix the surfactant concentration at 0.1CMC, dissolve Arg ₁₆ in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and add an equal volume of Arg ₁₆ buffer according to the charge ratio of polyelectrolyte to surfactant at 1:1. The solution, the surfactant and the oppositely charged polyelectrolyte construct a supramolecular assembly through hydrophobic and electrostatic interactions, mix evenly, and let stand for 15 minutes to obtain an assembly solution, and the weight percent concentration of the assembly solution is 0.01%;

Ⅳ Preparation of test solution:

Pipette the fluorescent probe organic solution prepared in step I again with a microsampler, and add them to the assembly solution obtained in III respectively, so that the final concentration of the probe in the assembly solution is 10 ^-6 mol L ^-1 . 70kHz exposed ultrasonic treatment for 30 minutes, and after standing for 1 hour, the test solution was obtained;

Ⅴ Determination of catalytic hydrolysis time:

Incubate the test solution obtained in step IV at 37°C for 15 minutes, dissolve trypsin in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and add the same volume of trypsin with different concentrations to the test solution to add The buffer solution was used as a blank control, the concentration ranged from 0.01 to 100 μg mL ^-1 , and the fluorescence spectrum tracking was carried out at 37°C. The ratio of the decrease of the fluorescence emission peak intensity was plotted against the time, and the fluorescence emission under different concentrations of trypsin catalyzed hydrolysis conditions was obtained. The time range in which the peak intensity decreases linearly;

Ⅵ Determination of trypsin activity:

According to the time range obtained in step V, the maximum value of the common linear time range under each concentration condition was taken, and the ratio of the decrease in fluorescence emission peak intensity was plotted against the concentration of trypsin added to obtain a detection standard curve.

The ratio of fluorescence intensity decrease is (II ₀ )/I ₀ ×100 %, I ₀ represents the fluorescence intensity at the fluorescence emission peak of the blank control with only buffer solution added, and I represents the fluorescence intensity at the emission peak after adding different concentrations of trypsin.

The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment: under the same test conditions, the same concentrations of alkaline phosphatase, lysozyme, glucose oxidase, and trypsin were added to the assembly solution, and the fluorescence spectrum was detected after incubation at 37°C for half an hour.

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Embodiment 3

Ⅰ Preparation of organic solution of fluorescent probe:

Using Sudan Red as a fluorescent probe, dissolve Sudan Red in chloroform, prepare a Sudan Red chloroform solution with a concentration of 10 ^-1 mol L ^-1 in a 10 mL volumetric flask, and further dilute to 10 ^-3 mol L ^-1 with chloroform ;

Ⅱ Determination of critical micelle concentration CMC of surfactant:

Sodium cetyl sulfate was dissolved in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and a solution of 10 ^-2 mol L ^-1 was prepared in a 10 mL volumetric flask, and further diluted with buffer solution to obtain Gradient solution with a concentration range of 1mol L ^-1 ~10 ^-7 mol L ^-1 ;

Use a microsampler to pipette the organic solution of the fluorescent probe prepared in step I, and add it to the prepared gradient solution, so that the final concentration of the fluorescent probe in the gradient solution is 10 ^-6 mol L ^-1 . Ultrasonic treatment was performed at 40 kHz for 60 minutes, and the fluorescence excitation spectrum was detected after standing for 12 hours.

The emission peak intensity of the fluorescence emission spectrum is plotted against the logarithmic value of the surfactant concentration, and an s-shaped curve is obtained by fitting, and the concentration corresponding to the first mutation point of the curve is the critical micelle concentration of the surfactant. The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Fix the surfactant concentration at 0.8 CMC, dissolve Arg ₁₂ in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and add an equal volume of Arg ₁₆ buffer solution according to the charge ratio of polyelectrolyte to surfactant at 1:1 , the surfactant and the oppositely charged polyelectrolyte construct a supramolecular assembly through hydrophobic and electrostatic interactions, mix evenly, and let stand for 10 minutes to obtain an assembly solution, the weight percent concentration of which is 0.12%;

Ⅳ Preparation of test solution:

Pipette the fluorescent probe organic solution prepared in step I again with a micro-sampler, and add them to the mixed solution obtained in III, so that the final concentration of the probe in the mixed solution is 10 ^-6 mol L ^-1 . Ultrasonic treatment was performed for 60 minutes, and the test solution was obtained after standing for 12 hours;

Ⅴ Determination of catalytic hydrolysis time:

Incubate the test solution obtained in step IV at 37°C for 10 minutes, dissolve trypsin in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and add the same volume of trypsin with different concentrations to the test solution to add The buffer solution was used as a blank control, the concentration ranged from 0.01 to 100 μg mL ^-1 , and the fluorescence spectrum tracking was carried out at 37°C. The ratio of the decrease of the fluorescence emission peak intensity was plotted against the time, and the fluorescence emission under different concentrations of trypsin catalyzed hydrolysis conditions was obtained. The time range in which the peak intensity decreases linearly;

Ⅵ Determination of trypsin activity:

According to the time range obtained in step V, the maximum value of the common linear time range under each concentration condition was taken, and the ratio of the decrease in fluorescence emission peak intensity was plotted against the concentration of trypsin added to obtain a detection standard curve.

The ratio of fluorescence intensity decrease is (II ₀ )/I ₀ ×100 %, I ₀ represents the fluorescence intensity at the fluorescence emission peak of the blank control with only buffer solution added, and I represents the fluorescence intensity at the emission peak after adding different concentrations of trypsin. The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment: under the same test conditions, the same concentrations of alkaline phosphatase, lysozyme, glucose oxidase, and trypsin were added to the assembly solution, and the fluorescence spectrum was detected after incubation at 37°C for half an hour.

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Embodiment 4

Ⅰ Preparation of organic solution of fluorescent probe:

Except that the fluorescent probe used is Coumarin 481, the others are the same as Embodiment 2;

Ⅱ Determination of critical micelle concentration CMC of surfactant:

Except that surfactant used is sodium dodecylsulfonate, other is with embodiment 2;

The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Except that the surfactant concentration is 1.6CMC, the polyelectrolyte used is Arg ₁₄ , and the weight percent concentration of the assembly solution is 2%, the others are the same as Embodiment 1;

Ⅳ Preparation of detection solution: same as embodiment 2;

Ⅴ Determination of catalytic hydrolysis time: same as embodiment 2;

Ⅵ Determination of trypsin activity: same as embodiment 1;

The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment:

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Embodiment 5

Ⅰ Preparation of organic solution of fluorescent probe:

Except that the fluorescent probe used is BODIPY FL, and the concentration is 10 ^-5 mol L ^-1 , the others are the same as Embodiment 2;

ⅡDetermination of critical micelle concentration CMC of surfactant:

Except that the surfactant used is sodium tetradecylsulfonate, and the final concentration of the fluorescent probe is 10 ^-8 mol L ^-1 , the others are the same as Embodiment 2;

The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Except that the surfactant concentration is 0.6CMC, the polyelectrolyte used is Arg ₁₃ , and the weight percent concentration of the assembly solution is 0.08%, the others are the same as Embodiment 1;

Ⅳ Preparation of detection solution: same as embodiment 2;

Ⅴ Determination of catalytic hydrolysis time: same as embodiment 2;

Ⅵ Determination of trypsin activity: same as embodiment 1;

The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment:

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Embodiment 6

Ⅰ Preparation of organic solution of fluorescent probe:

Except that the fluorescent probe used is BODIPY R6G, others are the same as embodiment 5;

ⅡDetermination of critical micelle concentration CMC of surfactant:

Except that surfactant used is sodium cetyl sulfonate, other is the same as embodiment 5;

The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Except that the surfactant concentration is 0.5CMC, the polyelectrolyte used is Arg ₁₅ , and the weight percent concentration of the assembly solution is 0.07%, the others are the same as Embodiment 1;

Ⅳ Preparation of detection solution: same as embodiment 2;

Ⅴ Determination of catalytic hydrolysis time: same as embodiment 2;

Ⅵ Determination of trypsin activity: same as embodiment 1;

The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment:

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Embodiment 7

Ⅰ Preparation of organic solution of fluorescent probe:

Except that the fluorescent probe used is BODIPY TMR, others are the same as embodiment 5;

ⅡDetermination of critical micelle concentration CMC of surfactant:

Except that the final concentration of the fluorescent probe is 10 ^-7 mol L ^-1 , the others are the same as Embodiment 2

The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Except that the surfactant concentration is 0.9CMC, the polyelectrolyte used is Arg ₁₁ , and the weight percent concentration of the assembly solution is 0.12%, the others are the same as in Embodiment 1;

Ⅳ Preparation of detection solution: same as embodiment 2;

Ⅴ Determination of catalytic hydrolysis time: same as embodiment 2;

Ⅵ Determination of trypsin activity: same as embodiment 1;

The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment:

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Embodiment 8

Ⅰ Preparation of organic solution of fluorescent probe:

Except that the fluorescent probe used is BODIPY 581/591, the others are the same as Embodiment 5;

ⅡDetermination of critical micelle concentration CMC of surfactant:

Except that the final concentration of the fluorescent probe is 10 ^-7 mol L ^-1 , the others are the same as Embodiment 3;

The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Except that the surfactant concentration is CMC, the polyelectrolyte used is Arg ₁₆ , the others are the same as in Embodiment 1, and the weight percent concentration of the assembly solution is 0.15%,;

Ⅳ Preparation of detection solution: same as embodiment 2;

Ⅴ Determination of catalytic hydrolysis time: same as embodiment 2;

Ⅵ Determination of trypsin activity: same as embodiment 1;

The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment:

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Embodiment 9

Ⅰ Preparation of organic solution of fluorescent probe:

Except that the fluorescent probe used is BODIPY TR, others are the same as embodiment 5;

ⅡDetermination of critical micelle concentration CMC of surfactant:

Except that the final concentration of the fluorescent probe is 10 ^-7 mol L ^-1 , the others are the same as Embodiment 4;

The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Except that the surfactant concentration is 1.2CMC, the polyelectrolyte used is Arg ₁₄ , the others are the same as in Embodiment 1, and the weight percent concentration of the assembly solution is 0.15%,;

Ⅳ Preparation of detection solution: same as embodiment 2;

Ⅴ Determination of catalytic hydrolysis time: same as embodiment 2;

Ⅵ Determination of trypsin activity: same as embodiment 1;

The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment:

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Embodiment 10

Ⅰ Preparation of organic solution of fluorescent probe:

Except that the fluorescent probe used is BODIPY 630/650, the others are the same as Embodiment 5;

II Determination of the critical micelle concentration CMC of the surfactant: same as embodiment 5;

The critical micelle concentration of the surfactant was obtained by fluorescence spectrometry.

Ⅲ Preparation of supramolecular assembly solution:

Except that the surfactant concentration is 0.4CMC, the polyelectrolyte used is Arg ₁₂ , and the weight percent concentration of the assembly solution is 0.05%, the others are the same as Embodiment 1;

Ⅳ Preparation of detection solution: same as embodiment 2;

Ⅴ Determination of catalytic hydrolysis time: same as embodiment 2;

Ⅵ Determination of trypsin activity: same as embodiment 1;

The detection results of fluorescence spectrum showed that the decrease ratio of fluorescence intensity at the emission peak position had a good linear relationship with the concentration of trypsin.

Specificity experiment:

The results of fluorescence spectroscopy showed that the assembly system had good specificity for the detection of trypsin, and other proteins would not affect the detection.

Claims (3)

1. A method for label-free fluorescent detection of trypsin, characterized in that it is a method based on supramolecular assembly disassembling under enzyme-catalyzed hydrolysis to realize trypsin detection, and its specific steps are: Ⅰ Preparation of organic solution of fluorescent probe: Use commercially available pyrene, Nile red, coumarin 481, Sudan red, boron fluorochrome BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPY TR or BODIPY 630/650 with a purity higher than 98% for hydrophobicity The dye is a fluorescent probe, and the hydrophobic dye is dissolved in an organic solvent of commercially available spectrum pure acetone, chloroform or methanol, and an organic solution with a concentration of 10 ^-3 ~ 10 ^-6 mol L ^-1 fluorescent probe is prepared; Ⅱ Determination of critical micelle concentration CMC of negatively charged alkyl sulfate or alkyl sulfonate: Dissolve negatively charged alkyl sulfate or alkyl sulfonate in phosphate buffer solution with a concentration of 10 mM and pH 8.0 to prepare a gradient solution with a concentration range of 1mol L ^-1 ~10 ^-7 mol L ^-1 , pipette the fluorescent probe organic solution prepared in step Ⅰ with a micro-sampler, and add it to the gradient solution so that the final concentration of the fluorescent probe in the gradient solution is 10 ^-8 ~10 ^-6 mol L ^-1 . 100kHz exposure ultrasonic treatment for 10-60 minutes, after standing for 0.5-12 hours, perform fluorescence spectrum detection to measure the critical micelle concentration CMC of negatively charged alkyl sulfate or alkyl sulfonate; Ⅲ Preparation of supramolecular assembly solution: According to the critical micelle concentration CMC of the negatively charged alkyl sulfate or alkylsulfonate that step Ⅱ obtains, determine that the negatively charged alkyl sulfate or alkylsulfonate concentration is 0.1～1.6CMC, will carry The oppositely charged peptide hydrolyzed by trypsin was dissolved in a phosphate buffer solution with a concentration of 10 mM and pH 8.0 to make a peptide solution; The sulfonate charge ratio is 1:1, add an equal volume of polypeptide solution catalyzed by trypsin hydrolysis to the solution of negatively charged alkyl sulfate or alkyl sulfonate, negatively charged alkyl sulfate or The alkyl sulfonate and the oppositely charged polypeptide hydrolyzed by trypsin form a supramolecular assembly through hydrophobic and electrostatic interactions, mix well, and let stand for 10 to 15 minutes to obtain a supramolecular assembly solution. The supramolecular assembly The weight percentage concentration of solution is 0.01～0.2%; Ⅳ Preparation of test solution: Pipette the fluorescent probe organic solution prepared in step I again with a microsampler, and add them to the supramolecular assembly solution obtained in step III, so that the final concentration of the fluorescent probe in the supramolecular assembly solution is 10 ^-8 ~10 ^-6 mol L ^-1 , treated with 40~100kHz exposure ultrasonic wave for 10~60 minutes, and left to stand for 0.5~12 hours to obtain the test solution; Ⅴ Determination of catalytic hydrolysis time: Incubate the test solution obtained in step IV for 10-15 minutes at 35-37°C, dissolve trypsin in a phosphate buffer solution with a concentration of 10 mM and pH 8.0, and add the same volume of trypsin with different concentrations to the test solution Solution, with only phosphate buffer solution added as a blank control, the final concentration of trypsin ranged from 0.01 to 100 μg mL ^-1 , and the fluorescence spectrum tracking detection was carried out at 35 to 37°C, and the ratio of the decrease in fluorescence emission peak intensity was compared with time. The figure shows the time range for the linear decline of fluorescence emission peak intensity under different concentrations of trypsin catalyzed hydrolysis conditions; Ⅵ Determination of trypsin activity: According to the time range obtained in step V, take the maximum value of the linear time range under the condition of different concentrations of trypsin from 0.01 to 100 μg mL ^-1 , and plot the ratio of the decrease of fluorescence emission peak intensity against the concentration of trypsin added to obtain the detection standard curve. 2. according to the method for a kind of label-free fluorescent detection trypsin described in claim 1, it is characterized in that described alkyl sulfate is commercially available analytical pure sodium lauryl sulfate, tetradecyl sodium sulfate or hexadecyl sulfate Sodium Alkyl Sulfate; Alkyl Sulfonate is commercially available analytically pure Sodium Dodecyl Sulfonate, Sodium Tetradecyl Sulfonate or Sodium Cetyl Sulfonate. 3. according to the method for a kind of label-free fluorescent detection trypsin described in claim 1, it is characterized in that the described polypeptide that is catalyzed and hydrolyzed by trypsin is commercially available purity higher than 99%, and the length is 10～16 band positive The charged arginine sequence Arg ₁₀ ~ Arg ₁₆ .

Images